The overall goal of this research is to determine how transfusion-induced iron overload affects hematopoietic stem cell transplant (HSCT) outcomes. HSCT is the only cure for multiple malignant and nonmalignant conditions; however, its use is limited by significant morbidity and mortality. Iron overload is common in patients undergoing HSCT, and in observational human studies, transfusion-induced iron overload is a risk factor for poor HSCT outcome. Our preliminary data implicates iron-mediated changes to gut microbiota as a modifiable therapeutic target in the peri-transplant period. Recent studies show that modulating the microbiome of mice and pediatric patients undergoing allogenic HSCT decreases the adverse effects associated with this treatment. Thus, to connect iron-mediated effects on gut microbiota with HSCT outcomes, we will expand upon our preliminary results to further characterize the effects of transfusion-induced iron overload on the bone marrow microenvironment and the gut microbiota. In Aim #1, we will test the hypothesis that transfusion-induced iron overload disrupts the bone marrow microenvironment by increasing reactive oxygen species (ROS) in the supporting cells of the niche. In Aim #2, we will test the hypothesis that the gut microbiota affect the response of the bone marrow niche to iron overload and hematopoietic recovery following HSCT. This will provide evidence for a relationship between iron and the gut microbiome in mediating HSCT outcome, and determining the contribution of specific microbial products to the relevant underlying mechanism(s). This research will lead to innovative and eminently translatable approaches for improving HSCT outcome in the setting of iron overload, in conjunction with improving our understanding of host-microbiome interactions.